Although it is widely accepted that B cells with self-reactivity are deleted or rendered functionally inactive, self-reactive antibodies, referred to as natural autoantibodies can be found in the serum of healthy animals. Such natural autoantibodies are produced by a small fraction of B cells with distinctive expression of the cell surface glycoprotein CD5. These CD5+ autoreactive B cells are more frequently generated from fetal B cell precursors as a part of B-1 B cell development than those in the adult bone marrow. Our research explores why such natural autoreactive B cells should exist, the mechanism whereby they develop, and their potential for dysregulation. By establishing and investigating mouse natural autoreactive B cell models expressing natural anti-Thy-1 autoantibody (ATA), we found that self-antigen is important for CD5+ ATA autoreactive B-1 B cell accumulation with relatively higher B cell receptor (BCR) signal intensity mediating a positive selection process. In contrast, negative selection occurs for cells expressing the identical BCR during conventional B cell development (B-2) in spleen from bone marrow. In this renewal, we will obtain a comprehensive understanding of the mechanism for B cell subset development. We will first investigate the mechanism of conventional non-autoreactive B-2 B cell maturation, in particular the significance of non-BCR signaling provided by other lymphocytes. We will assess the role of T cells and fetal B-1 B cells in adult B-2 (follicular B cell) development and immunity and their potential interaction (Aim 1). To understand why autoreactive B-1 positive selection occurs in relation to fetal B cell development, we will identify a fetal B-1 signature that we hypothesize reflects distinctive cellular machinery determining a difference in BCR signaling threshold, compared to bone marrow (B-2) development. To follow early-generated B-1 cell fate, we will produce Lysmd2-GFP reporter mice, based on our recent identification of this gene as a member of the fetal B-1 signature (Aim 2). Accomplishing the proposed work will significantly advance our understanding of autoreactive B-1 positive selection and B cell development in general. We hypothesize that early generation of natural autoreactive lymphocytes occurs for critical protective reasons, as a component of the innate immune system, to help development of the adaptive immune systems that follows. 7. Relevance to Public Health Abnormal expansions of CD5+ B cells can occur with age, in certain autoimmune mouse strains, and as B lymphomas. In humans, CD5 expression is a hallmark of late developing chronic B cell leukemia (B CLL), accounting for a third of adult leukemia cases in the United States and Western Europe. Arriving at a comprehensive understanding of B cell development, selection, and maturation mechanism(s), and establishing how CD5+ natural autoreactive B cell are normally regulated and function, as proposed in this application, will be critically important for designing rational therapies of such dysregulated CD5+ B cells.